Neurons in autonomic ganglia share many features but can be distinguished by their ganglionic morphology, location and neurotransmitter. Autonomic neurons are derived from the neural crest, a transitory population of precursor cells. The generation of neuronal diversity involves integration of extrinsic and intrinsic instructive cues that result in the neuron subtype-specific expression of transcriptional regulators. For autonomic noradrenergic (NE) neurons, bone morphogenetic 4 (BMP4) is an essential extrinsic cue. BMP4- mediated signaling recruits the basic helix-loop-helix DMA binding protein HAND2, in an NE cell type-specific manner, to a core network of transcriptional regulators expressed by autonomic neuron precursor cells. Both gain-of-function and loss-of-function studies demonstrated a necessary role for HAND2 in neurogenesis and cell type-specific gene expression required for the generation of NE neurons. The underlying mechanisms of HAND2 function remain unknown. The goals of this proposal are to: 1) unravel the mechanisms by which HAND2 contributes to neurogenesis of NE neurons, and 2) determine the mechanism by which HAND2 links expression of pan-neuronal genes to cell type-specific genes. We will use targeted deletion of HAND2 at early and late stages of neurogenesis to reveal the consequences to differentiation as neurons and expression of NE neurotransmitter due to loss of HAND2. Next, we will ectopically express HAND2 in precursor cells and identify target genes by expression profiling. Using promoter reporter constructs in transient transfection assays, we will examine functional interactions of HAND2 with identified target genes. Finally, we will examine intracellular signaling pathways downstream of BMP4 and dependent upon Smad1/4 translocation, MapK and PKA to understand how BMP4-mediated signaling influences the expression and function of HAND2. These studies are particularly compelling because HAND2 is markedly overexpressed in neuroblastoma, a solid pediatric tumor derived from neural crest cells destined to give rise to sympathetic ganglion neurons or adrenal medullary cells. These tumors are heterogeneous in their phenotype and can regress or lead to death. Since we will provide a detailed analysis of fundamental mechanisms underlying neuron cell fate choice and sub-type identity, our studies have the potential for development of novel treatment strategies for neuroblastoma based on genetic regulation of neurogenesis. [unreadable] [unreadable]